We wish to continue our studies on the "Function of the Subunits of Eukaryotic Chromosomes" by focusing attention on the relationship between nuclear organization and gene expression at the level of the chromosomal loop. For these studies we have selected the powerful biological system of immunoglobulin genes in the mouse for which we have considerable experience. OUr experimental approach combines biochemical, cell biological, immunological, molecular, and reverse genetic techniques. During the previous funding period of this grant we discovered a family of DNA sequences that organize chromosomal loops in the interphase nucleus which we termed MARs ("Matrix Association Regions"). Significantly, both MAR sequences and their nuclear binding sites are evolutionarily conserved, and MARs containing topoisomerase II sites are located in the immunoglobulin genes next to the enhancer elements. Here we propose to elucidate the functions of MARs, to determine the DNA sequences and proteins that are functionally important, and to localize MAR sequence binding sites within the nucleus. Specifically, we wish to address the four following Specific Aims: (1) To functionally characterize the role of MARs in gene transcription: . position requirements with respect to enhancers . capacity to override chromosomal position effects . ability to serve as boundaries between chromatin domains (2) To delineate the functionally crucial DNA sequences within a MAR (3) To characterize the proteins that bind to Mar sequences (4) To determine the nuclear location of MAR sequence binding sites